Non-vesicular GABA release via GABA transporter reversal

通过 GABA 转运蛋白逆转释放非囊泡 GABA

• 批准号: 7572873
• 负责人: GEORGE B RICHERSON
• 金额: $ 33.1万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7572873
• 关键词: 4-Aminobutyrate aminotransferase; Action Potentials; Address; Affect; Affinity; Anions; Anticonvulsants; Astrocytes; Behavior; Biological Assay; Brain; Calcium; Cell Culture Techniques; Cells; Chinese Hamster Ovary Cell; Complex; Coupled; Data; Dependence; Disease; Elements; Epilepsy; Equilibrium; Extracellular Fluid; FarGo; Figs - dietary; Floor; Frequencies; GABA transporter; Goals; Health; High Pressure Liquid Chromatography; Hippocampus (Brain); Ischemia; Knock-out; Knockout Mice; Lead; Link; Measurement; Measures; Mediating; Membrane; Membrane Potentials; Methods; Mus; Neuroglia; Neurons; Neurotransmitters; Physiological; Physiology; Play; Proteins; Regulation; Relative (related person); Rest; Ringer' s solution; Role; Seizures; Side; Signal Transduction; Simulate; Slice; Source; Stimulus; Stroke; Swelling; Synapses; Synaptic Transmission; Synaptic Vesicles; System; Testing; Theoretical model; Time; Vacuum; Vesicle; Vigabatrin; Wild Type Mouse; Work; design; excitotoxicity; extracellular; gamma-Aminobutyric Acid; in vivo; insight; mind control; nervous system disorder; neurotransmission; neurotransmitter release; neurotransmitter reuptake; novel; receptor; research study; response; reuptake; stoichiometry

DESCRIPTION (provided by applicant): The long-term goal of our work is to define the role of GABA transporters, which are a critical element of the GABAergic system that maintains brain excitability within normal limits. Many neuroscientists view GABA transporters simply as scavengers of GABA that has been released by vesicular fusion. However, new data suggest that the behavior of GABA transporters is much more complex, and that they play an active role in neuronal inhibition that goes far beyond simply reuptake of GABA. For example, among the neurotransmitter transporters they have a particularly low threshold for reversal, and when they reverse they release GABA into the extracellular fluid. Even when they don't reverse they play an important role in regulation of the amount of tonic inhibition, a newly discovered form of GABA signaling due to continuous activation of high affinity extrasynaptic GABAA receptors. Thus, accumulating evidence indicates that GABA transporters are not just GABA vacuum cleaners, but play a much more dynamic role in control of brain excitability. We have proposed the novel hypothesis that during neuronal firing the increase in membrane potential and rise in intracellular [Na+] leads to GABA transporter reversal, an increase in extracellular [GABA], and more tonic inhibition. We have further proposed that GABA transporters are one of the major determinants of extracellular [GABA] at rest, by virtue of the fact that they will only transport GABA into cells until they reach their equilibrium, and under normal conditions this equilibrium is reached when extracellular [GABA] is still relatively high. Thus, by establishing the "floor level" of extracellular GABA, they are responsible for maintaining a minimum amount of tonic inhibition. Here we plan experiments that test these hypotheses by: 1) Directly measuring how easily GAT1 and GAT3 reverse, using a novel, highly sensitive functional assay of transporter reversal; 2) Determining whether neurons can release GABA during action potentials via GAT1 reversal, 3) Measuring intracellular and extracellular [GABA] in response to treatment with the anticonvulsant vigabatrin, which selectively enhances tonic inhibition; 4) Determining the relative importance of GAT1 reversal compared to other forms of nonvesicular GABA release, and; 5) Defining the mechanism of a GAT1- independent nonvesicular form of GABA release that appears to come from glia. Loss of normal GABAergic inhibition can lead to seizures, and enhancement of inhibition may limit excitotoxicity during ischemia. Thus, the work proposed here will lead to better insight into normal synaptic physiology and control of inhibition during pathophysiological conditions such as epilepsy and strokes. The anticipated results may lead to new treatments for neurological disease aimed at enhancing nonvesicular GABA release and targeting the newly discovered form of tonic inhibition.

描述（由申请人提供）：我们工作的长期目标是确定GABA转运蛋白的作用，GABA转运蛋白是GABA能系统的关键要素，可将大脑兴奋性维持在正常范围内。许多神经科学家将GABA转运蛋白简单地视为囊泡融合释放的GABA的清道夫。然而，新的数据表明，GABA转运蛋白的行为要复杂得多，它们在神经元抑制中发挥着积极的作用，远远超出了简单的GABA再摄取。例如，在神经递质转运蛋白中，它们具有特别低的逆转阈值，并且当它们逆转时，它们将GABA释放到细胞外液中。即使它们不逆转，它们也在调节强直性抑制量方面发挥着重要作用，强直性抑制是由于高亲和力突触外GABAA受体的持续激活而新发现的GABA信号传导形式。因此，越来越多的证据表明，GABA转运蛋白不仅是GABA真空吸尘器，而且在控制大脑兴奋性方面发挥着更积极的作用。我们提出了一个新的假说，即在神经元放电过程中，膜电位的增加和细胞内[Na+]的升高导致GABA转运蛋白逆转，细胞外[GABA]的增加和更多的紧张性抑制。我们进一步提出，GABA转运蛋白是细胞外[GABA]在休息时的主要决定因素之一，因为它们只将GABA转运到细胞中，直到它们达到平衡，并且在正常条件下，当细胞外[GABA]仍然相对较高时，达到这种平衡。因此，通过建立细胞外GABA的“最低水平”，它们负责维持最低量的紧张性抑制。在这里，我们计划通过以下实验来验证这些假设：1）使用一种新型的、高度灵敏的转运蛋白逆转功能测定法直接测量GAT 1和GAT 3逆转的容易程度; 2）确定神经元是否可以通过GAT 1逆转在动作电位期间释放GABA; 3）测量细胞内和细胞外[GABA]对抗惊厥剂氨己烯酸治疗的反应，氨己烯酸选择性地增强强直抑制; 4）确定与其他形式的非囊泡GABA释放相比，GAT 1逆转的相对重要性，以及; 5）定义似乎来自神经胶质的非GAT 1依赖性非囊泡形式的GABA释放的机制。正常GABA能抑制的丧失可导致癫痫发作，抑制的增强可限制缺血期间的兴奋性毒性。因此，这里提出的工作将导致更好地了解正常的突触生理和控制抑制在病理生理条件下，如癫痫和中风。预期的结果可能会导致神经系统疾病的新治疗方法，旨在增强非囊泡GABA的释放，并针对新发现的强直抑制形式。